Devices and Methods for Nerve Regeneration

ABSTRACT

The present invention is directed to a nerve regeneration conduit including a resorbable tube having a matrix therein. The matrix is characterized by substantially parallel, axially aligned pores extending the length of the matrix. The matrix is formed by the axial freezing of a slurry having little or no significant radial thermal gradient during the freezing process. The matrix is used to bridge the gap between the severed ends of a nerve and provide a scaffold for nerve regeneration.

CROSS REFERENCE SECTION

This application claims priority to U.S. Patent Application No.62/429,363 entitled “Devices and Methods for Nerve Regeneration” andfiled on Dec. 2, 2016, which is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of mammalian nerveregeneration. Specifically, the present invention relates to methods ofmaking devices useful for nerve regeneration. The present inventionfurther relates to devices and methods that assist in regenerating asevered peripheral nerve by bridging the gap between the ends of thesevered peripheral nerve and providing a scaffold to support regrowth ofnerve tissue.

BACKGROUND

Nerve regeneration conduits are known. See U.S. Pat. No. 5,019,087.Methods for making nerve regeneration prostheses are also known. SeeU.S. Pat. No. 4,955,893 and US Patent Application Publication No.2011/0129515. However, it is desirable to promote regeneration of nervetissue to rejoin the ends of severed nerves.

It is therefore an object of the present invention to provide aregenerative scaffold to enhance axon and Schwann cell propagationduring the process of peripheral nerve regeneration across nerve gapsgreater than allowed by prior entubulation repair techniques. It is alsoan object of the present invention to provide an apparatus and processthat allows the routine manufacture of a biocompatible nerveregeneration conduit comprising a resorbable tube filled with aresorbable matrix having controlled pore size and parallel, axiallyoriented pore alignment resembling the Schwann cell basal lamina.

It is also an object of the present invention to provide acollagen-based conduit filled with a resorbable matrix that includesinductive macromolecules to enhance the regenerative potential of theconduit. Such a capability has the potential for enhancing regenerativecapacity for large gap defects from injuries or other trauma. These andother objects, features, and advantages of the invention or certainembodiments of the invention will be apparent to those skilled in theart from the following disclosure and description of exemplaryembodiments.

SUMMARY

Embodiments of the present invention are directed to nerve guides andare further directed to devices and methods for tissue regeneration and,in particular, nerve tissue regeneration using a scaffold of the presentinvention. According to certain aspects of the present invention, amethod is provided in which axial freezing of a suspension, dispersionor slurry (collectively “slurry”) having little or no significant radialthermal gradient followed by freeze drying results in a matrix having aplurality of passages, channels, pathways or pores (collectively,“pores”) generally spanning one end of the matrix to the other. In oneembodiment of the method, the slurry is thermally insulated to provideit with little or no significant radial thermal gradient during theaxial freezing of the slurry. The terms dispersion, slurry andsuspension are used interchangeably herein.

The configuration of pores produced by methods of the present inventionthat span one end of the matrix to the other promotes the growth oftissue, and more specifically nerve tissue, into and through the matrixas a whole, as physical obstructions within the pores in the matrix areminimized by the method of the present invention. The pores can beaxially-oriented to the extent that the pores allow nerve tissue to growinto and through the matrix. The pores can be axially-oriented that spanone end of the inner matrix to the other and the outer collagen-basedtube or conduit can be configured with pores that span one end of theouter tube or conduit to the other. Such pores are directed along theaxis of the severed nerve and promote the growth of the nerve tissueinto and through the pores of the matrix scaffold.

According to one aspect of the present invention, the matrix allowsnerve tissue to grow from opposite ends of the matrix and join togetherat a point within the matrix, as a characteristic of a plurality ofpores is that they span one end of the matrix to the other in anunobstructed manner. In an exemplary embodiment, a severed nerve can bereconnected by interconnecting each severed end of the nerve with thematrix of the present invention and allowing nerve tissue to growthrough the matrix from opposite ends until contacting and combiningtogether and, preferably, forming a functioning nerve where it was oncesevered and nonfunctioning. In this manner, embodiments of the presentinvention include a prosthesis or implant or scaffold to regeneratedamaged nerve fibers that have a gap or distance between the severedends of the nerve fibers. The prosthesis or implant or scaffold orconduit can be made to have different diameters and/or lengths, asdesired, for use with different diameter nerve and different gaps ordistances between severed nerves. The prosthesis or implant or scaffoldor conduit of the present invention are also capable of showingincreased fatigue resistance following cyclic compression.

According to embodiments of the present invention, the step ofmaintaining the suspension at little or no significant radial thermalgradient while axially freezing the suspension followed by freeze dryingoptimizes the formation of axially oriented pores. The greater thenumber and consistency of axially oriented pores within the matrix fromone end of the matrix to the other, the greater the ability of thematrix to allow, and even promote, nerve growth therein andtherethrough.

According to alternate embodiments of the present invention, the matrixis useful as a device by itself, and can be combined with one or moreconnectors such as cuffs to allow the joining of the matrix torespective ends of a severed nerve. According to a different embodiment,the matrix can be preformed and then inserted into and housed by ahollow conduit. Alternatively, a suspension can be introduced into aconduit or hollow conduit/tube which is maintained at little or nosignificant radial thermal gradient and then the suspension can beaxially frozen followed by freeze drying to form the matrix within theconduit/tube. In either embodiment, the conduit housing the matrix canlikewise be combined with one or more connectors, such as cuffs to allowthe joining of the matrix to respective ends of a severed nerve. Stillalternatively, the end portions of the conduit may be hollow, may lackmatrix or otherwise may extend beyond the matrix therein to allow thesevered end of a nerve to be inserted into the conduit in a manner tocontact the matrix and allow nerve growth therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrograph at 15× of a cross-section of amatrix produced by the method of the present invention.

FIG. 2 is a scanning electron micrograph at 100× of a cross-section of amatrix produced by the method of the present invention.

FIG. 3 is a scanning electron micrograph at 150× of a longitudinal crosssection of a matrix produced by the method of the present invention.

FIG. 4 is an image showing various sizes of prototype conduitsfabricated, including hollow and filled conduits/collagen tubes.

FIGS. 5A-D are scanning electron micrograph images of the hollowconduit/collagen tube. The cross-section shown in FIG. 5A highlights thepore structure of the conduit. The abluminal surface shown in FIGS. 5Band 5C has pore structures which are very irregular whereas the luminalsurface, seen on the right side of the last image, FIG. 5D, has no poresdetectable.

FIGS. 6A-D are scanning electron micrograph images of the filled conduitproduced by the isopropanol freezing method. The cross-sections, FIGS.6A and 6B, and longitudinal-sections, FIGS. 6C and 6D, demonstrate theinteraction between the pore structure of the outer conduit/collagentube and internal matrix.

FIG. 7 is a graphical analysis of the internal matrices of the conduitprototype. The more pixels present at 90°, the more aligned the poresare axially. Pixels close to 0° and 180° represent pores that areperpendicular to the length of the conduit and therefore not alignedaxially.

FIG. 8 is a graphical comparison of displacement vs. force of conduitunder compressive load between the 1st cycle and 100th cycle.

FIG. 9 is a graphically depicts typical stress vs time of a conduitunder compressive load for 100 cycles.

FIG. 10 illustrates S42 cells cultured on well-plates coated with arange of macromolecules for 7 days. The cells were stained with glialfibrillar acid protein (GFAP-red) characteristic of Schwann cells andHoechst (nuclei-blue).

FIG. 11 depicts DNA content assessed after 7 days' culture of S42Schwann cell line on well plates coated with a range of macromolecules.# denotes p<0.01 statistical significant difference between CSFL orCSFL2 and all other groups.

FIG. 12 depicts the effect of the combination of all 3 macromolecules onDNA content assessed after 7 days' culture of S42 Schwann cell line. #denotes p<0.01 statistical significant difference between CSFLL2 and allother groups.

FIG. 13 illustrates Schwann cell migration from sciatic nerve explantsinto the filled conduits after 7 days' culture. The red cells showpositive staining for S100b. The nuclei were stained blue with Hoechst.

FIG. 14 illustrates dorsal root ganglia cultured on filled conduits (CS)for 7 days. Red staining is positive for axons and Schwann cells. Nucleiof cells are stained blue with Hoechst.

FIGS. 15A-B illustrate dorsal root ganglia cultured on filled conduitswith internal matrices composed of CSFL (FIG. 15A) and CSFL2 (FIG. 15B)for 7 days. Red staining is positive for axons and Schwann cells. Nucleiof cells are stained blue.

FIG. 16 depicts a potential set up for a direct freeze-drying method.The system includes a polystyrene block with multiple molds, stainlesssteel screws, or other thermally conducting plug, pole, etc.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Embodiments of the present invention are based on the discovery of adevice and method that produces axially aligned pores in a matrix usinga slurry having little or no significant radial thermal gradient duringaxial freezing of the slurry. In one embodiment, the slurry is thermallyinsulated to maintain little or no significant radial thermal gradientduring axial freezing. The term “little or no significant radial thermalgradient” means no detectable thermal gradient in the radial directionof the slurry or at the very least any thermal gradient that exists doesnot adversely affect the formation of axially aligned pores. Forpurposes of exemplary embodiments of the present invention, little or nosignificant radial thermal gradient is achieved by insulating the slurryduring axial freezing of the slurry. Without being bound by anyscientific theory, axial freezing of the slurry having little or nosignificant radial thermal gradient is believed to produce axiallyaligned ice crystals along the length of the slurry. Freeze dryingremoves the crystals and leaves behind a matrix having axially alignedpores.

In accordance with a first aspect of the invention, the slurry used toform the matrix includes materials known to those of skill in the artused to form such matrices. The materials include biocompatible and/orbioresorbable materials that can form a liquid slurry or suspension.Such materials or macromolecules include collagen, laminin, fibronectin,merosin (also referred to as laminin-2), hyaluronic acid, chitin,chitosan, keratin, polyglycolic acid, polylactic acid, cellulose and thelike. The materials can be used alone or in combination with each other.It is to be understood that the list of materials is not exhaustive andthat one of skill in the art will readily identify other materialsuseful to make slurries based on the present disclosure. In certainexemplary embodiments, the matrix is formed from collagen. Collagen is afibrous protein and constitutes the major protein component of skin,bone, tendon, ligament, cartilage, basement membrane and other forms ofconnective tissue. Collagen is biodegradable, and when implanted in thebody, is absorbed at a rate that can be controlled by the degree ofintra- or intermolecular cross-linking imparted to the collagen moleculeby chemical or physical treatment. Thus upon implantation, the collagenmatrix can be designed to be absorbed as the tissue grows into thematrix, such as when nerve tissue regenerates and grows into the matrix.

In certain exemplary embodiments, the matrix includes collagen and atleast one glycosaminoglycan. Exemplary glycosaminoglycans includechondroitin sulfate, (chondroitin-6-sulfate proteoglycan), dermatansulfate, keratin sulfate, hyaluronic acid, and the like. Theglycosaminoglycans can be used alone or in combination with each other.It is to be understood that the list of glycosaminoglycans is notexhaustive and that one of skill in the art will readily identify othermaterials useful to make slurries based on the present disclosure. Incertain exemplary embodiments, the collagen and the glycosaminoglycanare cross-linked. Cross-linking can be achieved by heating under vacuumor by treatment with chemical cross-linking agents, e.g.,glutaraldehyde, formaldehyde, chromium sulfate, carbodiimide, adipyldichloride, and the like.

The materials to form the matrix are combined with a liquid to form aslurry which is then introduced into a mold to form a matrix of adesired shape. Suitable liquids within the scope of the invention shouldbe capable of being removed by freeze drying and include water, andaqueous fluids containing alcohol, acetic acid and the like. The liquidscan be used alone or in combination with each other. It is to beunderstood that the list of liquids is not exhaustive and that one ofskill in the art will readily identify other materials useful to makeslurries based on the present disclosure. Methods of freeze dryingmaterials that contain liquids such as water are known to those of skillin the art.

Other materials can be included into or otherwise form the slurry andtherefore can be incorporated into the matrix as desired. Such othermaterials include drugs, growth factors, extracellular matrixcomponents, fibrous materials and the like. The other materials can beused alone or in combination with each other. It is to be understoodthat the list of other materials is not exhaustive and that one of skillin the art will readily identify still other materials based on thepresent disclosure.

Other materials that can be included or otherwise form the slurryinclude inductive macromolecules from the basal lamina. The inductivemacromolecules play a significant role in the nerve regeneration processin terms of soliciting guidance, proliferation axonal growth andremyelination (Wallquist et al., 2005, Bunge et al., 1986, Buttery andffrench-Constant, 1999). The term inductive macromolecules can include,but is not limited to glycosaminoglycans such as hyaluronans,chondroitin sulfate, dermatan sulfate, heparin sulfate, keratin sulfate,proteoglycans such as aggrecan, decorin, syndecans, as well as collagen,elastin, fibronectin, laminin-1 and laminin-2, and other growth factors.Preferably, fibronectin in combination with laminin-1 and laminin-2 hasshown an unexpected and surprising synergy capable of promoting Schwanncell migration and growth, as well as axonal growth from dorsal rootganglions (DRGs). In certain exemplary embodiments, the inductivemacromolecules are cross-linked. Cross-linking can be achieved byheating under vacuum or by treatment with chemical cross-linking agents,e.g., glutaraldehyde, formaldehyde, chromium sulfate, carbodiimide,adipyl dichloride, and the like.

It is to be understood that molds of any desired shape can be used inthe present invention based upon the principles disclosed herein and foruse where it is desired that tissue grow into the pores of the matrix.Particular molds can be used to produce various desired matrix shapesincluding tubes, cylinders, rectangles, spheres, sheets and otherdesired shapes and can even be in the same general shape as wound sitesor tissue defects so that the matrix can be fit to the particular woundsite or tissue defect. Although the matrices of the present inventioncan be used to regenerate and connect severed nerves, the matrices alsohave other uses where reconnecting severed or broken tissue orrebuilding damaged tissue through tissue regeneration would beadvantageous. Such applications include regeneration of tendon,articular cartilage, bone, corticospinal tracts, and other linearlyaligned structures.

An exemplary mold can produce a single matrix or a mold may be capableof producing a plurality of matrices, such as where the mold includes aplurality of chambers of desired design into which the slurry can beintroduced and then subject to axial freezing. Such a mold contemplatescommercial manufacture of the matrices of the present invention where itis desired to mass produce such matrices in a batch format. Individualchambers within a mold are insulated so the slurry therein has little orno significant thermal gradient during axial freezing. Each individualchamber can include an insulating material therein surrounding thechamber or the entire mold can be formed from an insulating materialthereby insulating the slurry in the individual chambers. Suitableinsulating materials include STYROFOAM, AEROGEL and the like.

According to an additional exemplary embodiment, the matrix can be usedby itself or it can be preformed and then inserted into a hollow conduitfor use as a prosthesis. Alternatively, the matrix can be formeddirectly inside a hollow conduit. According to this embodiment, a slurryis placed into a hollow conduit or outer tube which is insulated. Theaxial freezing process and freeze drying process takes place to form thematrix within the conduit. The conduit containing the matrix is thenused as a prosthetic device to connect severed tissue. In oneembodiment, the conduit is biodegradable or bioresorbable. An exemplaryperiod of time for biodegradability or bioresorbability is within about1 to about 3 months. An exemplary embodiment is a resorbable collagentube, as is commercially available under the brand name NEURAGEN fromIntegra LifeSciences Corporation, Plainsboro, N.J. Methods for makingcertain exemplary embodiments of the resorbable tube are disclosed inU.S. Pat. No. 5,019,087, which is incorporated herein by reference inits entirety.

In certain exemplary embodiments, the collagen in the resorbable tube isType I collagen, and the tube further comprises a laminin-containingmaterial. Laminin is a glycoprotein that is an abundant component of allbasement membranes. As used herein, the phrase “laminin-containingmaterial” is meant to include purified laminin itself or a materialwhich contains laminin and other basement membrane components and iscapable of forming a dispersion from which the resorbable tubes aremade. Materials which contain laminin include basement membranes, humanplacenta, and an extract of a mouse sarcoma known in the art asMatrigel. In certain exemplary embodiments, the amount of Type Icollagen that is combined with the laminin present in thelaminin-containing material on a dry weight basis is in the ratio ofabout 90:10 to 40:60. In certain exemplary embodiments other optionaladditives which may aid in the nerve regeneration may also be present inthe resorbable tube in addition to collagen, for example, heparin,heparan sulfate proteoglycan, glycosaminoglycans such as hyaluronicacid, chondroitin sulfate and others, growth hormones such as epidermalgrowth factor (EGF), nerve growth factor, glycoproteins such asfibronectin, and the like. The glycosaminoglycans can be used alone orin combination with each other. It is to be understood that the list ofoptional additives is not exhaustive and that one of skill in the artwill readily identify other optional additives useful in the presentinvention based on the present disclosure.

In certain exemplary embodiments, the resorbable tube may becross-linked. This can be done with chromium sulfate, formaldehyde,glutaraldehyde, carbodiimide, adipyl dichloride, and the like. The rateat which the resorbable tube of the present invention is resorbed invivo in a mammal is dependent on the degree of cross-linking. Factorscontrolling the extent of crosslinking are the type and concentration ofthe cross-linking agent, the pH, and the temperature of incubation. Incertain exemplary embodiments, the nerve regeneration conduits of thepresent invention are cross-linked to such an extent that they arecompletely resorbed within about 1 to about 3 months.

In certain exemplary embodiments, the resorbable tube/outer tube orconduit has a length of about 1 cm to about 15 cm, and an inner diameterin the range of from about 1 mm to about 1.5 cm. The length of theresorbable tube/outer tube or conduit may vary with the length of thenerve gap to be bridged, and the inner diameter may vary with thediameter of the nerve. In certain exemplary embodiments, the resorbabletube/outer tube or conduit has a length of about 1 cm to about 15 cm or2 cm to about 10 cm, e.g., a length of about 2 cm to about 4 cm, alength of about 3 cm to about 7 cm. In certain exemplary embodiments,the inner diameter of the resorbable tube/outer tube or conduit is inthe range from about 1 mm to about 15 mm, e.g., from about 1.5 mm toabout 10 mm or about 1.5 mm to about 5.0 mm. The wall thickness of theresorbable tube represents a balance between desired permeability andenough compressive strength to prevent collapse. Preferably, the tubesare made as thin as possible while still withstanding suturing andcollapse when used in vivo. In certain exemplary embodiments, theresorbable tube has a wall thickness in the range of from about 0.2 mmto about 1.2 mm, e.g. about 0.1 mm to about 0.8 mm. In certain exemplaryembodiments, the resorbable tube is less porous than the matrix.

According to another exemplary embodiment of the present invention, anouter collagen tube is formed. The outer collagen tube or conduit ishollow. According to certain exemplary embodiments, the collagen in theresorbable tube or conduit is Type I collagen. According to anotherexemplary embodiment, the collagen in the resorbable tube or conduit isalkali-treated (AT) collagen.

According to another exemplary embodiment, the tube further comprises alaminin-containing material. In certain exemplary embodiments, theamount of Type I collagen that is combined with the laminin present inthe laminin-containing material on a dry weight basis is in the ratio ofabout 90:10 to 40:60.

According to another exemplary embodiment, the collagen tube is filledwith a slurry to form an internal matrix. In certain exemplaryembodiments, the slurry includes collagen, chondroitin sulfate, and oneor more inductive macromolecules. Preferably, the inductivemacromolecules include fibronectin, laminin-1, or laminin-2. In certainexemplary embodiments, the amount of fibronectin, laminin-1, orlaminin-2 combined on a dry weight basis is in the ratio of about 1:1 iftwo macromolecules are used. In certain exemplary embodiments, theamount of chondroitin sulfate, fibronectin, laminin-1, and laminin-2combined on a dry weight basis is in the ratio of about 1:1:1:1. Incertain exemplary embodiments, the concentration of chondroitin sulfate,fibronectin, laminin-1, and laminin-2 is about 5 μg/ml for eachcomponent. In certain exemplary embodiments, the concentration ofchondroitin sulfate, fibronectin, laminin-1, and laminin-2 is about 1-5μg/ml for each component. In certain exemplary embodiments, theconcentration of chondroitin sulfate, fibronectin, laminin-1, andlaminin-2 is about 5-10 μg/ml for each component. In certain exemplaryembodiments, the concentration of chondroitin sulfate, fibronectin,laminin-1, and laminin-2 is about 1-10 μg/ml for each component. Incertain exemplary embodiments, the slurry further includes aglycosaminoglycan such as dermatan sulfate, keratin sulfate, orhyaluronic acid, or any combination thereof. According to exemplaryembodiments of the present invention, the outer collagen tube with theinternal matrix described above forms a conduit that forms asurprisingly synergistic microenvironment that enhances Schwann cellsurvival.

According to exemplary embodiments of the present invention, the slurryis subject to axial freezing. A cooling gradient is generated in theaxial direction of the slurry when in the mold by rapid heat transferfrom the slurry to a cooling medium, and where the cooling gradient hassubstantially no radial component. Cooling in this manner formssubstantially parallel, axially aligned ice crystals in the slurry.According to certain exemplary embodiments, a heat sink at one end ofthe slurry causes heat to be drawn out of the slurry in an axial manner.A heat sink in accordance with the principles of the present inventionincludes a thermally conducting plug that contacts the slurry and inturn is in contact with a cooling medium. The thermally conducting plugacts as a heat sink, as well as sealing off one end of the tube whereslurry is added to the tube. The plug can be made of any material thathas high thermal conductivity, such as metals and metal alloys (e.g.,brass, steel, copper, zinc, nickel, and aluminum, among others). Thethermally conducting plug can be inserted into the mold that containsthe slurry, thereby contacting the slurry. According to one embodiment,the thermally conducting plug can serve as a stop within the mold, suchas when the mold shape is a cylinder and is positioned perpendicular tothe cooling medium with the thermally conducting plug directlycontacting the cooling medium. According to another exemplaryembodiment, the slurry that is used to fill the hollow outer collagentube or conduit is frozen along an axial direction of the slurry, withthe slurry having no detectable radial thermal gradient, to form afrozen slurry having parallel axially aligned crystals. According toanother exemplary embodiment, the step of freezing includes contactingthe proximal end of the outer collagen tube or conduit to a heat sinkwith the slurry freezing from a proximal end of the slurry to a distalend of the slurry to form the frozen so that a slurry cooling gradientis generated in the axial direction of the slurry when in the mold byrapid heat transfer from the slurry to a cooling medium, and where thecooling gradient has substantially no radial component.

Suitable cooling media include any solid or liquid media capable offreezing the liquid slurry, for example, a cooling medium that maintainsa temperature between about −78° C. and about −196° C. Certain exemplaryembodiments of the method of the invention include the step ofcontacting the thermally conducting plug with a cooling medium toprovide a cooling gradient in the axial direction of the insulated tube.The cooling medium may be at least one of liquid nitrogen, dry ice, anisopropanol/dry ice mixture, and silicone oil cooled by liquid nitrogen,and the like, whether directly contacting the thermally conducting plugor indirectly through a different media such as a cold plate and thelike. The cooling media can be used alone or in combination with eachother. It is to be understood that the list of cooling media is notexhaustive and that one of skill in the art will readily identify othercooling media useful to freeze slurries based on the present disclosure.Once the slurry, such as an aqueous slurry, is completely frozen, thetube filled with frozen aqueous slurry is dried under vacuum (e.g., byfreeze drying or lyophilizing) to produce a nerve regeneration conduitof the present invention.

As heat is drawn out of the slurry in an axial manner by the heat sink,freezing of the slurry proceeds along the length of the slurry from theend of the slurry proximal to the heat sink to the distal end of theslurry. The slurry, whether directly in a mold or in a conduit, isthermally insulated with little or no significant thermal gradient tosignificantly affect freezing of the slurry at the point of contact ofthe slurry with the wall of the mold or the conduit.

Axial freezing combined with thermal insulation followed by freezedrying produces substantially parallel, axially aligned pores extendingthe length of the matrix. Ice crystal formation occurs along thegradient of cooling. If the temperature gradient is uniform through avolume of space, and each plane in the volume perpendicular to thedirection of the temperature gradient is of a uniform temperature, andthe gradient is sufficient to propagate ice crystal formation throughoutthe length of the gradient, then the formation of ice crystals in such adefined region will extend through the region in a manner aligned to thedirection of the gradient. Thus, the ice crystals will be substantiallyparallel to the gradient and substantially parallel to each other. Incertain exemplary embodiments of an aqueous slurry and a tubular mold,this axial cooling gradient is achieved by thermally insulating theaqueous slurry or the walls of the tube containing the aqueous slurry tobe frozen. Insulating the tube can be done with any material thatprevents heat transfer through the wall of the tube. This substantiallyeliminates any radial component in the cooling gradient, providing auniform axial cooling gradient. Subsequent freeze drying or lyophilizingof the frozen slurry in the tube results in a matrix having openchannels comprising substantially parallel, axially oriented pores.

According to another exemplary embodiment of the present invention, theouter collagen tube and the slurry used to fill the tube are subject todirect freeze-drying. The collagen tube and the slurry are directfreeze-dried along an axial direction of the slurry. According toanother exemplary embodiment, the slurry has no detectable radialthermal gradient during direct freeze-drying and forms a frozen slurryhaving parallel axially aligned crystals. According to another exemplaryembodiment, the direct freeze-drying of the collagen tube and the slurryremoves the parallel axially aligned crystals and forms the internalmatrix that has parallel axially aligned outer tube/conduit pores andinternal matrix pores that span one end of the matrix to the other.Accordingly, the direct freeze-drying of the outer collagen tube withthe internal matrix forms a conduit that forms a microenvironment thatenhances Schwann cell survival.

The pores of the matrix, internal matrix, and the outer tube or conduithave an average diameter of about 10 μm to about 300 μm, about 50 μm toabout 250 μm, about 75 μm to about 200 μm, 50 μm to about 150 μm, 50 μmto about 80 μm, about 100 μm to about 200 μm, or about 50 μm to about250 μm. According to another exemplary embodiment, the pores have anaverage diameter of about 5 μm to about 360 μm or about 160 μm to about180 μm. The matrix can be any length depending upon the desiredapplication, however, suitable lengths include from about 2 cm to about20 cm, from about 3 cm to about 15 cm or from about 5 cm to about 10 cmand ranges therebetween.

In certain exemplary embodiments, the resorbable tube/outer tube orconduit has an abluminal surface with an irregular surface porestructure. The pores of the abluminal surface have an average diameterof about 20 μm to about 200 μm, about 40 μm to about 180 μm, about 60 μmto about 160 μm, 80 μm to about 140 μm, 100 μm to about 120 μm, about100 μm to about 200 μm, or about 50 pin to about 150 μm.

In accordance with a certain aspect of the invention, the matrix of thepresent invention is used to promote in vivo regeneration of a severedmammalian nerve so as to bridge a gap between a first end and a secondend of the severed nerve. The matrix can be included within a conduithaving a first end and a second end. The matrix may be flush with thefirst end and second end of the conduit or it may be recessed within oneor both ends of the conduit. Alternatively, the matrix may extend beyondone or both ends of the conduit, if desired. According to oneembodiment, connectors may be used to connect the conduit containing thematrix, or the matrix alone, to the severed end of a nerve. Suitableconnectors within the scope of the present invention overlap the conduitand the nerve and include wraps or cuffs with or without sutures or anyother suitable connector design that can be used to connect the conduitor matrix to the severed end of a nerve.

One particular example of a connector is a collagen sheet or wrap thatcan be placed or wrapped around the nerve and the conduit and thensecured in place, such as by using sutures. One such collagen sheet orwrap is marketed under the NEURAWRAP mark from Integra LifeSciencesCorporation, Plainsboro, N.J. Such sheets or wraps can be in acylindrical form having a longitudinal slit where opposing ends of thewrap can be pulled apart, the nerve inserted and then the wrap canrebound into a cylindrical position around the nerve. Such sheets orwraps can be made from biodegradable or bioerodable materials such ascollagen, laminin, fibronectin, merosin, hyaluronic acid, chitin,chitosan, keratin, polyglycolic acid, polylactic acid, cellulose and thelike. The materials can be used alone or in combination with each other.It is to be understood that the list of materials is not exhaustive andthat one of skill in the art will readily identify other materialsuseful to make sheets or wraps based on the present disclosure.

Where the matrix is flush with an end of the conduit, another suitableconnector is a cuff having a first open end and a second open end. Thecuff has an outer diameter larger than the outer diameter of theconduit. The cuff conforms to the outer shape of the conduit. Forexample, if the conduit is tubular or cylindrical, the cuff will be atubular or cylindrical cuff. A cuff is placed onto the outer end of theconduit with a portion of the cuff extending over the end of theconduit. The first end of the severed nerve is inserted into the cuffextension and is brought into contact with the first end of the matrixto form a first junction between the severed nerve and the conduit. Thenerve, cuff and conduit are all secured in place at this first junctionaccording to methods known to those skilled in the art, such assuturing. The second end of the severed nerve is brought into contactwith the second end of the matrix to form a second junction between thesevered nerve and the conduit. This junction may also be secured inplace with a cuff as described above. Cuffs according to embodiments ofthe present invention can be formed from various materials includingcollagen laminin, fibronectin, merosin, hyaluronic acid, chitin,chitosan, keratin, polyglycolic acid, polylactic acid, cellulose and thelike. The materials can be used alone or in combination with each other.It is to be understood that the list of materials is not exhaustive andthat one of skill in the art will readily identify other materialsuseful to make cuffs based on the present disclosure. Suitablecommercially available cuffs include resorbable collagen tubes having alength sufficient for a cuff, as are commercially available under thebrand name NEURAGEN from Integra LifeSciences Corporation, Plainsboro,N.J. Methods for making certain exemplary embodiments of the resorbabletube are disclosed in U.S. Pat. No. 5,019,087, which is incorporatedherein by reference in its entirety.

If, according to an exemplary embodiment, the matrix is recessed withinone or both ends of the conduit, i.e. the end of the conduit extendspast the matrix therein, the severed nerve is introduced into theconduct until it contacts the matrix to form a junction and the nerve issecured in place within the conduit using methods known to those skilledin the art, such as suturing. No connector such as a sheet, wrap or cuffis required with this exemplary embodiment, although a sheet, wrap orcuff could still be used if desired.

In certain exemplary embodiments where the ends of the conduit, such asa resorbable tube, extend past matrix therein, a distance into each endof the tube is unfilled with the slurry to form the matrix, oralternatively, matrix is removed from within the tube, or stillalternatively, matrix of length shorter than the tube is inserted intothe tube. Methods for making an embodiment where the matrix is formedwithin the tube include plugging the bottom of the tube with a plug thatextends a distance into the tube, followed by filling the tube withslurry up to a desired point, which can include the end of the tube or alocation before the end of the tube thereby allowing the end of the tubeto extend past the matrix. These embodiments allow insertion of asevered nerve end into a hollow end of the resorbable tube and contactof the nerve end with the matrix inside the tube. The nerve end insidethe hollow end of the resorbable tube may then be sutured to the tube.

In a certain exemplary embodiment, nerve regeneration conduits accordingto the present invention include a resorbable tube having a matrix ofcontrolled pore size and parallel structure that mimics Schwann cellbasal lamina, which significantly enhances Schwann cell migration andaxon regeneration through the conduit. Schwann cells are nonneuronalcellular elements that provide structural support and insulation toaxons. Thus, by using the conduits of the present invention in a mannersuch that the respective ends of a severed nerve are brought intocontact with each end of the conduit fashioned from a resorbable tubefilled with a matrix having substantially parallel, axially alignedpores extending the length of the matrix, greater numbers ofregenerating axons are stimulated, many of which become mylenated, asubstantial increase in the initial rate of the outgrowth of fibers andmylenated axons is produced, and the regenerating axons are able to spanthe gap between the severed nerve by growing through the matrix.

The matrices of the present invention promote parallel axial alignmentof regenerated nerve tissue accompanied by a large number of Schwanncells. The axial oriented pores of the matrices of the present inventionpromote peripheral nerve regeneration that is axially aligned to thedirection of the resorbable matrix, with the oriented pores beingsubstantially parallel to each other along the entire length of thedesired route of nerve regeneration. For example, an axon entering apore of the nerve regeneration conduit of the present invention shouldexit the opposite end of the conduit at substantially the same relativeposition.

The pore size and parallel alignment in the matrix are intended toresemble the Schwann cell basal lamina so as to encourage axon growth.In certain exemplary embodiments, the population of pores in the matrixhave an average diameter of about 10 μm to about 300 μm. In an alternateembodiment, the population of the pores in the matrix have an averagediameter from about 40 μm to about 180 μm or an average diameter fromabout 80 μm to about 120 μm.

Certain exemplary embodiments of the present invention are directed to asystem including an apparatus used to freeze the slurries of the presentinvention. The apparatus includes a vessel filled with a heat transferfluid and including a coil through which coolant flows. The temperatureof the fluid is monitored and controlled at a desired temperature. Acollagen tube of desired size is fitted at one end with a heat sink suchas a copper rod and the collagen tube is inserted into PVC tubing. ThePVC tubing with the collagen tube and copper rod is housed in insulatingmaterial with the heat sink protruding beyond the insulating material.According to one method of the present invention, a slurry is pouredinto the collagen tube and the heat sink is contacted to the heattransfer fluid, i.e. liquid cooling medium, with the insulating materialacting as a float, as the insulating material is buoyant when placed inthe liquid cooling material. In this embodiment the collagen tube isheld in a substantially vertical position within the insulating materialwith the heat sink contacting the liquid cooling material. The slurry isallowed to freeze and the frozen slurry is then lyophilized to form amatrix. The matrix may then be further processed such as by undergoingcrosslinking. The matrix may then be packaged and sterilized accordingto methods known to those of skill in the art.

Certain other exemplary embodiments of the method of the presentinvention include surrounding a bioresorbable or biodegradable tube havea thermally conducting plug at one end with a material that thermallyinsulates the tube and the slurry within so that there is substantiallyno thermal gradient in the radial direction of the tube. As anon-limiting example, the tube may be inserted into a block of polymerfoam, polystyrene block, or other insulating material, such as STYROFOAMor AEROGEL, so that the plug protrudes from the bottom of the block andthe open end of the tube is flush with the top of the block. The blockof polymer foam serves as a thermal insulator to prevent heat transferthrough the walls of the tube. Thus, any thermal gradient in the tubewill have substantially no radial component. The polymer foam maycomprise one or more of the following insulating materials: polystyrene,polyurethane, polyethylene, ceramic and silicone, and the like. Theinsulating media can be used alone or in combination with each other. Itis to be understood that the list of insulating media is not exhaustiveand that one of skill in the art will readily identify other insulatingmedia based on the present disclosure. Alternatively, the tube may beinsulated with any other material that substantially prevents heattransfer, for example, a vacuum- or gas-filled jacket or flask.

In certain exemplary embodiments, the method of using matrices,especially matrix-filled conduits, includes the steps of bringing therespective ends of the severed nerve into contact with each end of thenerve regeneration conduit of the present invention, which conduit isequal to or longer than the gap to be bridged so that no tension isplaced upon the severed nerve. Both the distal and proximal nerve endsare partially inserted into the ends of the resorbable tube, optionallyuntil the nerve ends contact the matrix filling the tube, and suturedover their perineuerium.

Use of the nerve regeneration conduit of the present invention promotesnerve regeneration across nerve gaps of up to 15 cm. In certainexemplary embodiments, the nerve regeneration conduit promotesregeneration across nerve gaps of about 2 cm to about 10 cm, e.g., about2 cm to about 4 cm, about 3 cm to about 7 cm. Nerve diameters that canbe accommodated by the nerve regeneration conduit of the presentinvention range from about 1 mm to about 1.5 cm, e.g., about 2 mm andabout 7 mm, about 3 mm to about 10 mm.

EXAMPLES

The following examples are specific embodiments of the present inventionbut are not intended to limit it.

Example 1

A resorbable tube having a porous matrix according to the presentinvention was achieved by highly controlled freezing and insulation ofan aqueous dispersion of collagen, followed by freeze drying. The porestructure and orientation of the matrix formed by the process of theinvention was examined by taking scanning electron microscope (SEM)images of transverse and longitudinal cross-sections of the resultingmatrix.

Specifically, a brass bolt was inserted into one end of a NEUROGENcollagen tube to both plug the tube and act as a heat sink. The tube wasthen inserted into a polystyrene foam block so that the bolt protrudedfrom the bottom face of the block, and the open end of the tube wasflush with the top face of the block. An aqueous collagen slurry wasprepared according to the method described in U.S. Pat. No. 6,969,523hereby incorporated by reference herein in its entirety. Suitablemethods to prepare a slurry useful in the present invention are alsodescribed in U.S. Pat. No. 5,997,895 hereby incorporated herein byreference in its entirety. The tube was filled with the aqueous collagenslurry, and the polystyrene foam block was floated with the bolt on thebottom face of the block immersed in a bath of silicone oil that hadbeen cooled by liquid nitrogen. The collagen slurry in the tube frozeentirely within about 30 minutes. The tube with the frozen slurry wasthen placed in a pre-cooled lyophilizer and freeze dried under vacuum.

FIG. 1 is a scanning electron micrograph at 15× of a cross-section of amatrix produced by the method of the present invention. FIG. 2 shows thematrix at 100×. FIG. 3 is a scanning electron micrograph at 150× of alongitudinal cross section of a matrix produced by the method of thepresent invention.

FIG. 1-FIG. 3 show substantially uniform pore structures where the poresare substantially parallel, axially aligned, and extending the length ofthe matrix. The pores are believed to mimic the highly axially orientedpore structure of Schwann cell basal lamina.

Example 2

A 4.5 cm long by 7 mm diameter tube was provided having walls formedfrom collagen and a laminin-containing material according to the processof U.S. Pat. No. 5,019,087. A copper rod was inserted into one end ofthe tube to both plug the tube and act as a heat sink. The tube was theninserted into a polystyrene foam block so that the copper rod protrudedfrom the bottom face of the block, and the open end of the tube wasflush with the top face of the block. The tube was filled with aqueousslurry of collagen and a glycosaminoglycan, and the polystyrene foamblock was floated with the copper rod on the bottom face of the blockimmersed in a bath of liquid nitrogen. The collagen slurry in the tubefroze entirely within 12 minutes. The tube with the frozen slurry wasthen placed in a pre-cooled lyophilizer and freeze dried under vacuum.The resulting porous matrix inside the tube was examined in bothcross-section and longitudinal section which showed a matrix havingsubstantially parallel, axially oriented pores extending the length ofthe matrix. Average pore diameter was between about 165 μm to about 180μm. The openness of the parallel pores was tested by inducing chargedfluorescent beads to migrate through the porous matrix under theinfluence of a voltage gradient. The majority of the charged fluorescentbeads were able to pass through the porous matrix from one end to theother, thus demonstrating that the majority of the parallel, axiallyaligned pores were open and extended along the length of the matrix.

Example 3

A tube was provided having walls formed from collagen and alaminin-containing material according to the process of U.S. Pat. No.5,019,087. A copper rod was inserted into one end of the tube to bothplug the tube and act as a heat sink. The tube was then inserted into apolystyrene foam block so that the copper rod protruded from the bottomface of the block, and the open end of the tube was flush with the topface of the block. The tube was filled with an aqueous slurry ofcollagen and a glycosaminoglycan made according to the method describedin Example 1, and the polystyrene foam block was placed in a Styrofoambox partially-filled with dry ice so that the copper rod directlycontacted the dry ice. The collagen slurry in the tube froze entirelywithin 1 hour 30 minutes. The tube with the frozen slurry was thenplaced in a pre-cooled lyophilizer and freeze dried under vacuum. Theresulting porous matrix inside the tube was examined in bothcross-section and longitudinal section which showed a matrix havingsubstantially parallel, axially oriented pores extending the length ofthe matrix. Average pore diameter was about 361 μm. The average porediameter resulting from the freezing method of this Example is notablylarger than the average pore size resulting from the freezing method ofExample 2, as it is believed that a slower freezing time allows forgrowth of larger ice crystals, which results in larger pores.

Example 4

An average freezing point of −22.03° C. was experimentally determinedfor an aqueous slurry of collagen and glycosaminoglycan by freezing fivesamples according to the table below.

Freezing Temperature Sample Weight (mg) (° C.) 1 15.0 −15.95 2 21.3−24.04 3 33.1 −24.14 4 31.4 −24.07 5 22.6 −21.94

Example 5

Conduit Lab-scale Production. The conduit is composed of a hollowcollagen tube and an internal matrix. The collagen conduit, or outerhollow tube, is composed of alkali-treated (AT) collagen. The collagenis stored at −20 C. The hollow collagen conduit is produced by making aslurry in the following manner. Cut small pieces from the frozen blockof collagen and weigh out 1.18 g into a glass bottle. Prepare 0.5Mlactic acid in distilled water at store at room temperature. Place theAT collagen into 100 ml lactic acid (0.5M). Leave the collagen todissolve in lactic acid overnight at 4° C. The following day, preparethe large blender and a box full of ice. Pour the AT collagen into abeaker and place on the box of ice. Blend at 15,000 rpm and monitor howwell the collagen is dispersing. This should be completely dispersed inabout 10-15 minutes. Make sure that the collagen is always cool to avoiddenaturation. Use a vacuum filter that has been modified with 100 and200 mesh. Use the 100 mesh first the filter the slurry. Once the slurryhas passed through, repeat the same thing with the 200 mesh. Store theslurry at 4° C.

Prepare mandrels using stainless steel rods and PTFE tape. Use the tapeto wrap around the stainless steel rods with enough tape to create thedesired diameter (1.5/3/5 mm). Measure the mandrels with verniercallipers to ensure the desired diameter has been created. Clean themandrels as well as the glass/plastic vials for spinning the collagenusing 0.05M acetic acid. Prepare 2.5M NaOH. Prepare 0.3% formaldehyde.Set up the dental drill and ensure that the speed is set toapproximately 7.5. Attach the mandrels onto the dental drill. Place 10ml of slurry into a vial. Add approximately 1.5-1.6 ml NaOH (2.5M) intothe slurry—add a few drops at a time. Shake vigorously or use a vortexmaking sure that the top is covered. The slurry should turn cloudy soonafter which will indicate that the collagen has precipitated. Make surethat the precipitation is homogenously distributed. Check to pH andensure that it is between 5.4 and 6. This volume should be enough tomake a conduit of 1.5 mm internal diameter, 5 cm in length. The ends ofthe conduit may have to be trimmed off. Place the mandrel attached tothe drill into the vial containing the precipitated collagen. Spin themandrel and make sure that the collagen is adhering onto the mandrel—dothis for approximately 10 seconds. Subsequently, spin the mandrel on theinner edge of the vial so that the collagen is compressed onto themandrel—do this for a further 10-20 seconds. The vial should now containa clear suspension with no collagen visible.

Remove the mandrel from the vial and use the glass plates to compressthe collagen further. Make sure that a glass spacer is between the glassplates. Place the mandrel between the glass plates making sure that thebottom end of the mandrel sits on the glass spacer so that the collagendoes not spin out of the mandrel. Spin the mandrel at the samespeed—7.5, for approximately 30 seconds. Detach the mandrel from thedental drill and place into falcon tubes containing 0.3% formaldehyde tocross-link the conduit for 90 minutes at room temperature. Subsequently,replace the formaldehyde with dH₂O and wash 3 times. Place the mandrelinto the dental drill again and spin between the glass plates tocompress the collagen further for approximately 20 seconds.Subsequently, place the mandrels in a freezer at −20° C. making surethat they are spaced well from each other. Leave the mandrels in thefreezer overnight. The following day, place the mandrels containing thecollagen on a stainless steel tray and freeze-dry at a final temperatureof −40° C. Once the collagen has been freeze-dried, carefully removethem from the mandrels making sure not cause tears on the collagen. Thiscan be done by spraying the collagen with 100% ethanol, then removeexcess ethanol by placing the mandrels on kimwipes/tissue. This shouldallow the collagen conduits to easily slide out of the mandrels withlittle damage caused. Allow the conduits to air dry overnight at roomtemperature.

Example 6

Internal matrix slurry production. The internal matrix is made from aslurry of 0.5% microfibrillar collagen, 0.044% chondroitin sulfate (fromshark cartilage). Prepare 0.05M glacial acetic acid Weigh 0.6 gmicrofibrillar collagen and place in a beaker containing 100 ml aceticacid (0.05M). Weigh 0.053 g chondroitin sulfate and place in a beakercontaining 20 ml acetic acid (0.05M). Leave the collagen and chondroitinsulfate in the fridge at 4 C overnight to dissolve. The following day,place the collagen in a box of ice and blend (15,000 rpm) initially forapproximately 30 minutes and then slowly add the chondroitin sulfateusing a syringe and tubing, a few milliliters at a time while blending.Blend for a further 10 minutes and subsequently de-gas to remove the airfrom the slurry. Place the slurry in a fridge at 4 C until needed butensure that it is de-gassed again before using.

For the incorporation of macromolecules (fibronectin and/orlaminin-1/-2) directly into the suspension, the desired concentration(initially chosen as 25 μg/ml) was made in a solution of PBS andsubsequently incorporated into the slurry. The slurry was then mixedwith a magnetic stirrer for 30 minutes and subsequently de-gassed asbefore. Soak-loading could also be utilized as a method of incorporationby soaking a suspension of the macromolecules onto the internal matrixof a freeze-dried conduit using a syringe. This was done forapproximately 1 hour at room temperature before re-freeze-drying at −40°C. final temperature.

Example 7

Filled conduit production. The filled conduit is produced using a 2-stepprocess. The conduits are filled with the internal matrix slurry andthen frozen using dry ice. The frozen conduits are then freeze-dried andcross-linked to produce the final product. Make sure that the internalmatrix slurry is fully de-gassed. Place a hollow conduit into PTFEtubing with a stainless steel screw (heat sink) attached on one end.Allow approximately 5 mm of space between the conduit and stainlesssteel screw. Add the slurry into the PTFE tubing containing the conduitwith a syringe and needle and allow the conduit to stand at roomtemperature for approximately 30 minutes. Subsequently, add more slurryinto the conduit making sure to eliminate all air bubbles from the PTFEtubing using the syringe and needle. Place the PTFE tubing containingthe conduit into a polystyrene block with a hole of the same diameter asthe tubing. Make sure that the stainless steel screw protrudes out ofthe polystyrene block and the open end of the tubing does not protrudeout i.e. make sure that the tubing is fully covered in the polystyrene.

The freezing process should be done in a flow-hood. Usingthermo-insulating gloves, place dry ice into a polystyrene box (approx.2 liters in volume). Pour isopropanol carefully into the dry ice. Thisshould bubble up so make sure that a safety face mask is utilized. Placethe polystyrene block containing the tubing with the conduit onto thedry ice and allow it to float making sure that the stainless steelscrews are fully submerged in the isopropanol. The internal matrixwithin the conduit should freeze fully within 45 minutes if thepolystyrene block is offers good insulation. Use an infra-redthermometer to ensure that the internal matrix has frozen (to below −20°C.) before removing the polystyrene block from the dry ice. Make surenot to interrupt the polystyrene block through movement so as to permitoptimal directional freezing. Once frozen, remove the PTFE tubing fromthe polystyrene block and place in a box for storage at −80° C.overnight or until one is ready to freeze-dry. Pre-cool the freeze-drierto −40° C. under a vacuum of approximately 300 Torr. When the shelvesreach the desired −40 C, release the vacuum and quickly open thefreeze-drier and place the PTFE tubing containing the conduit andinternal matrix on a stainless steel tray and onto one of the shelves.Freeze-dry to a final temperature of −40° C. and sublimate at 0° C.

Subsequently, remove the conduits from the PTFE tubing carefully usingdry forceps. See FIG. 4. Place the filled conduits in the vacuum oven at105° C. for 24 hours to sterilize and cross-link. For long-term studies,cross-link the filled conduits in sterile 0.3% formaldehyde andsubsequently wash 3 times with dH₂O.

Example 8

Direct freeze drying. This method was developed to eliminate themultiple steps required for the fabrication of the filled conduit. Asbefore, the conduit was placed into the PTFE tubing and filled with theslurry of the internal matrix. The tubing was also placed into thepolystyrene block as before but rather than freezing with dry ice, thepolystyrene block was placed directly onto the shelf of thefreeze-dryer. Set up the freeze-dryer so that the cooling rate is 1°C./min to a final temperature of −40° C. Sublimate the sample at 0° C.for 17 hours. This method will only work if the slurry is de-gassedthoroughly. The stainless steel screws must also have good contact withthe freeze-dryer shelf. This could be solved by using a stainless steeltray filled with water so that the screws are submerged in the water. Amold was designed and can be used to simplify the process further. SeeFIG. 16.

Example 9

The mechanical assessment of the conduit was carried out using suturepull-out tests and cyclic compression tests. Morphological assessment ofconduit structure and pore sizes was based on analyzing SEM images. Themethods used for these tests are described below.

This assessment was carried out to elucidate the ability of the conduitsto hold a suture under loads exceeding those anticipated in the intendeduse environment and therefore determine the strength of the conduit withrespect to the break strength of a suture. Nylon sutures of sizes10-0/9-0/8-0/7-0/6-0 were obtained for the assessment of suture pull-outstrength. A 10 mm conduit was hydrated for 1 hour in PBS at roomtemperature. One end of the conduit (2-5 mm from the end) was thenfirmly held by a specimen clamp attached to the bottom platen of theZwick testing machine. A suture (10-0/9-0/8-0/7-0/6-0) was then passedthrough the other end of the conduit (1 mm or 2 mm from the end) andtied to the top platen of the Zwick testing machine. The machine was setup so that the cross-head speed was 2.5 mm/min under tension. Sampleswere tested and the end point was determined as—when either the suturebroke or the conduit tore apart. The suture breakage or conduit tear ledto a decrease in force which automatically stopped the machine fromrecording of resistance to tension. The table below outlines the suturepull-out test data.

Suture Suture Conduit Force Suture Suture Conduit Force Size 10-0 BreakBreak (N) Size 9-0 Break Break (N) 1 x 0.75 1 x 0.95 2 x 0.72 2 x 0.91 3x 0.68 3 x 0.97 4 x 0.61 4 x 0.89 5 x 0.88 5 x 0.84 6 x 0.85 6 x 1.02 7x 0.89 7 x 1.1 8 x 0.77 8 x 0.94 9 x 0.73 9 x 0.93 10 x 0.78 10 x 0.9611 x 0.62 11 x 1.07 12 x 0.63 12 x 0.98 13 x 0.71 13 x 1.04 14 x 0.74 14x 1.02 15 x 0.76 15 x 0.94 16 x 0.58 16 x 1.15 17 x 0.61 17 x 1.08 18 x0.69 18 x 1.17 19 x 0.67 19 x 1.09 20 x 0.75 20 x 1.11 Suture SutureConduit Force Suture Suture Conduit Force Size 8-0 Break Break (N) Size7-0 Break Break (N) 1 x 1.25 1 x 1.56 2 x 1.47 2 x 1.62 3 x x 1.31 3 x1.73 4 x 1.14 4 x 1.54 5 x 1.56 5 x 1.77 6 x 1.49 6 x 1.79 7 x 1.38 7 x1.85 8 x x 1.31 8 x 1.51 9 x 1.42 9 x 1.89 10 x 1.21 10 x 1.95 11 x 1.1911 x 1.85 12 x 1.54 12 x 1.72 13 x x 1.43 13 x 1.88 14 x x 1.57 14 x1.66 15 x 1.27 15 x 1.92 16 x 1.48 16 x 1.78 17 x 1.44 17 x 1.74 18 x1.26 18 x 1.48 19 x 1.36 19 x 1.67 20 x 1.39 20 x 1.78 Suture SutureConduit Force Size 6-0 Break Break (N) 1 x 1.65 2 x 1.79 3 x 1.98 4 x1.63 5 x 1.74 6 x 1.71 7 x 1.93 8 x 2.04 9 x 1.74 10 x 2.06 11 x 1.68 12x 1.89 13 x 2.11 14 x 2.04 15 x 1.76 16 x 1.88 17 x 1.81 18 x 1.70 19 x2.19 20 x 1.96

The data collected from the suture pull-out tests showed that theconduit could resist tearing when sutures of sizes 10-0 and 9-0 wereapplied under tension. 8-0 sutures resulted in approximately 50% suturebreakage or conduit tear signifying that this is the limit in suturesize for the prototype conduits. 7-0 and 6-0 sutures both led toconduits tearing. The table below summarizes the data. “TEAR” signifiesthat the suture or conduit tore apart after a tensile load was applied.“X” signifies that the suture or conduit did not tear apart after atensile load was applied. “TEAR/X” signifies that approximately 50% ofconduits tore or sutures broke apart after a tensile load was applied.

SUTURE SIZE (DIAMETER) SUTURE CONDUIT 10-0 (0.02 mm)  TEAR X 9-0 (0.03mm) TEAR X 8-0 (0.04 mm) TEAR/X TEAR/X 7-0 (0.05 mm) X TEAR 6-0 (0.07mm) X TEAR

The conduits were tested to determine their ability to resist repeatedcompression from surrounding tissues. To achieve this, the conduits weretested for compression resistance and rebound force in a cycliccompression environment. A 10 mm conduit was hydrated for 1 hour in PBSbefore testing was carried out. The conduit was then placed on the lowerplaten (attached to a transparent trough) of the Zwick machine ensuringthat it was lying at the center of the platen. The upper platen was thenlowered so that the conduit was held between the 2 platens. The troughwas then filled with PBS ensuring that the upper platen was visiblebelow the water level. The Zwick machine was set up so that the conduitcompressed to 50% of its original diameter at a rate of 30 mm/min in acyclic manner. 100 cycles were run on each sample. The load cell of theZwick machine recorded the force required to compress the conduit in thecompression cycle and the rebounding force exerted by the conduit duringthe relaxation cycle. See FIGS. 8 and 9. It was evident that theconduits were capable of rebounding with minimal displacement of theirdiameters (FIG. 8). There was also minimal change in their forcerequired to compress the conduits when we compared the first andhundredth cycles of compression (FIG. 9). Importantly, the samplesmaintained over 90% of their original rebound force after 100 cycles.This highlights their high degree of fatigue resistance in compressiveenvironments.

Example 10

The conduits were imaged using SEM to determine the pore architectureand interaction between the hollow conduit and internal matrix. Both thehollow and filled conduits were sliced at twoorientations—cross-sections and longitudinal sections. In some cases,the internal matrix was produced alone, without the hollow conduit, soas to determine the degree of alignment of pores. These samples werealso sliced into cross-sections and longitudinal sections. Pore sizeanalysis was carried out manually using ImageJ. Clamp a 2 cm×2 cm pieceof PTFE tape onto the workbench. Place the sample or conduit onto thetape and hold in place with forceps to prevent movement. Using a newWilkinson Sword razor blade, slowly slice the sample into the requiredorientation making exaggerated slicing movements so that the blade isworked from one end of the sharp edge to the other. Having sliced thesample as required, use the same blade to detach it from the PTFE tape,rather than using forceps which can cause tears. Place the samples intovials for storage at room temperature. The table below includes the rawdata from the pore size analysis on SEM images. See FIGS. 5A-D and FIGS.6A-D.

Cross-section (um) Ablumen (um) Lumen (um) 1 106.667 139.402 2 105.409139.402 3 149.071 65.217 4 189.62 52.894 5 109.949 34.783 6 129.443215.206 7 160.278 181.623 8 109.949 27.498 9 152.315 44.339 10 203.08722.17 11 137.437 25.352 12 127.366 21.739 13 147.573 35.053 14 126.66730.744 15 116.619 29.166 16 121.655 26.087 17 133.5 25.352 18 124.54419.444 19 132.832 23.414 20 173.845 23.414

Example 11

Pore alignment was assessed using a method based on a Matlab programstemming from the paper Meng Sun et al., 2015 (DOI:10.1371/journal.pone.0131814) that assesses the pixel orientation of animage and gives a quantitative measure for this. The orientation iscalculated in degrees. Pixel counts at 90 degrees represent parallelalignment whereas less/greater than 90 degrees represent deviation fromalignment. The higher the count at 90 degrees therefore suggests greateralignment. FIG. 7 is a graphical analysis of the internal matrices ofthe conduit prototype. As discussed above, the more pixels present at90°, the more aligned the pores are axially. Pixels close to 0° and 180°represent pores that are perpendicular to the length of the conduit andtherefore not aligned axially. The fast_quantification_alignment.m isthe main function that imports the image data. The image should eitherbe in 8-bit or 16-bit. Change line 18 in the M.file‘fast_quantification_alignment.m’ if using an 8-bit or 16-bit image asfollows; blksze=15; thresh=0.5; thr=45 (for 8-bit) or thr=350 (for16-bit). For optimal assessment, ensure that all the images are taken atthe same magnification, resolution, contrast and of the same dimensions(x,y).

Example 12

In vitro assessment to determine the effect of the macromolecules oncellular response was carried out using a Schwann cell line (S42) thatwere purchased, as well as sciatic nerve explants and dorsal rootganglia that were isolated from rat tissue within the TERG labs. Thedesired macromolecules were coated on 6-well plates to assess theresponse of S42 Schwann cells. The S42 Schwann cells were then seededonto the 6-well plates at a density of 5×104 cells per well and themorphology and proliferation of the cells was assessed at 24/48 hoursand 7 days respectively. Results indicate that the PLL group led to highcell spreading, but limited proliferation after 7 days. In contrast, thecollagen coated groups led to greater cell spreading. Moreover, thepresence of fibronectin and/or laminin-1 or -2 led to cells displayingmore spindle-shaped morphologies which are characteristic of nativeSchwann cells. The presence of collagen and collagen-chondroitin sulfateled to over 3-fold increase in cell density compared to PLL after 7 days(FIG. 11). Moreover, when collagen was combined with fibronectin andlaminin-1/-2, this led to even further increases by approximately 50% incell density. The highest cell density was found in the CSFL2 grouphighlighting the potency of laminin-2 in cell proliferation.

TREATMENT GROUPS PLL Polylysine (50 μg/ml) (control) C Collagen (50μg/ml) CS Collagen-chondroitin sulfate (collagen-50 μg/ml, cs-5 μg/ml)CSF Collagen-chondroitin sulfate-fibronectin (collagen-50 μg/ml, cs-5μg/ml, fibronectin-5 μg/ml) CSL Collagen-chondroitin sulfate-laminin1CSL2 Collagen-chondroitin sulfate-laminin2 CSFL Collagen-chondroitinsulfate-fibronectin-laminin1 CSFL2 Collagen-chondroitinsulfate-fibronectin-laminin2 CSFLL2 Collagen-chondroitinsulfate-fibronectin-laminin1-laminin2 Total macromoleculeconcentration - 5 μg/ml i.e. for a mixture of fibronectin & laminin1 -2.5 μg/ml each so that there is a protein ratio of 1:1.

The same principle applies in terms of the ratio of macromolecules (1:1)incorporated if two proteins were utilized. However, the concentrationof the internal matrix within the conduit was chosen initially as 25μg/ml. There is some optimization required to be carried out before afinal concentration is selected.

Dorsal root ganglia were cultured on three conduit types with internalmatrices (CS, CSFL, CSFL2) for 7 days and assessed for Schwann cell andaxonal growth within the conduits. It was evident that there was axonaloutgrowth from the DRGs into the conduits by 7 days. There was alsoevidence of Schwann cell migration from the DRGs into the conduit. Thepresence of the axially aligned pores within the conduits permittedaxons to sprout along the length of the pores thereby highlighting theimportance of the physical guidance cues in the regenerative process.

It was evident that the conduits supported both Schwann cell and axonalgrowth into the internal matrices of the conduits. However, thecomposition influenced the depth of axonal penetration. Compared to thecontrol, which represents the basic composition (collagen-chondroitinsulfate), the presence of the additional inductive macromolecules(fibronectin in combination with laminin-1/-2) led to enhanced axonallength. In particular, the conduits containing fibronectin and laminin-1supported greater axonal penetration compared to the control conduit.The greatest axonal penetration was found in the conduits containingfibronectin and laminin-2 after 7 days (50% increase in cell density).See FIG. 10, FIG. 11, FIG. 14, and FIGS. 15A and 15B. Further, it wasevident that there was an additive effect on cell proliferation as aresult of the treatment with all 3 macromolecules. See FIG. 12. Therewas approximately 50% increase in proliferation between CSFL2 and CSFLL2(p<0.01), and approximately 150% increase between CSFL and CSFLL2(p<0.01). This demonstrates that combining all three macromolecules mayhave therapeutic potential in the early stages of the repair process byenhancing proliferation of Schwann cells.

The method to isolate the dorsal root ganglia follows. This protocol canbe utilized for newly born or adult rats and is done under terminalanesthesia. Lay the rat on its side and, under a dissecting microscope,cut off the head at the cervical flexure and the tail just caudal to thehind limbs using micro-dissecting scissors. Remove the ventral (belly)portion of the rat to isolate the dorsal (back) structures containingthe spinal cord. Position tissue dorsal side down and carefully removeany remaining viscera from the posterior wall. Place one blade ofmicro-dissecting scissors between vertebral column and spinal canal atrostral end and very carefully cut through vertebral column proceedingcaudally, then tease apart the right and left halves of vertebral columnto expose spinal cord and DRGs. Gently attempt to lift spinal cord fromdorsal structures by grasping cord at rostral end while carefully“cutting” behind and around DRGs to sever adherent tissues. Afterisolating all spinal cords, pluck off DRGs using dissecting forceps andtransfer to another 6-cm dish with ice-cold PBS. If nerve roots arepresent, they should be snipped away after removing DRGs from cords.Place the conduits, either sliced into cross-/longitudinal-sections oras whole conduits into a 24 well plate. Place the DRGs directly ontoconduits. Culture with DMEM-F12 media supplemented with 10% FBS and 1%penicillin/streptomycin.

Sciatic nerve explants were sliced into approximately 2 mm segments andcultured directly onto the filled conduits. Assessment of cell migrationfrom the conduits was subsequently carried out. The sciatic nerveexplant can be seen at the top of FIG. 13 and the blue stainingrepresents the nuclei of migrating cells from the sciatic nerve explantsafter 7 days. There was evidence of Schwann cells within the conduit asdemonstrated by the positive red staining thereby demonstrating theability of the conduit to permit Schwann cell migration from theexplants. Comparing the three conduit type (CS, CSFL, CSFL2), there wasno difference in the migration capacity of Schwann cells from theconduits.

The method used to isolate the sciatic nerve tissue is described next.This procedure is done under terminal anesthesia. Shave the fur from therat and place the animal on a surgical board in the prone position andsecure the front and hind legs to the board. Using a scalpel, make anincision on the leg of the rat. Separate the skin from the muscle andfascia. Slowly separate the interval between the biceps femoris and thesuperficial gluteal muscles with scissors until the sciatic nerve isexposed. Cut back the muscle along the skin incision line so that themajority of sciatic nerve is visible. Use scissors to cut off the nerveon both ends. Place the nerve on a petri-dish containing ice-cold PBS.Transfer to a tissue culture flow hood and cut the nerve into 2 mmsegments and culture onto the conduits/desired samples with high glucoseDMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

Samples were stained using immunofluorescence in the following describedmethod. Fix samples in 5% formalin for 1 hour and replace the formalinwith PBS for storage overnight. Prepare the blocking buffer with 3% FBS,0.3% Triton-X made up in PBS. Prepare dilution buffer with 1% FBS, 0.1%Triton-X made up in PBS. Wash the samples 3× in PBS. Incubate thesamples in the blocking buffer for 30-60 minutes at room temperature. Donot wash the samples after the blocking buffer has been incubated. Applythe primary antibody made up in the dilution buffer at the desiredconcentration (Rabbit anti-rat Neurofilament 1:50; Mouse anti-rat S100β1:50; Mouse anti-rat GFAP 1:40). Incubate at 4° C. overnight and thenwash in PBS. Apply the secondary antibody made up in the dilution bufferat the desired concentration (Goat anti-mouse IgG Alexa448 1:500; Goatanti-rabbit IgG Alexa546 1:500). Incubate for 1 hour at 4° C. and thenwash in PBS. Incubate in Hoechst 33342 at the desired concentration (1μg/ml) diluted in PBS to stain for the nucleus. Store the samples at 4 Cif not required immediately and ensure to cover in tin foil. See FIG.10, FIG. 14 and FIGS. 15A and 15B.

Cell proliferation was determined by PicoGreen DNA assay. ReagentsRequired: Quant-iT PicoGreen dsDNA assay Molecular Probes P7589, 1%Triton-X buffer. Remove the 6-well plates from the incubator and apply1% Triton-X and scrape off cells using a cell-scraper thoroughly. Placethe solution into eppendorfs. Prepare a 1×TE solution from the 20× stockprovided in the Quant-iT Kit (for each sample need 1.2 ml of 1×TE, forall standards need 6 ml of 1×TE). Prepare dilute PicoGreen solution,200-fold dilution of DMSO stock (20 μl of PicoGreen in 3.98 ml of 1×TEis sufficient for standards and 5 samples, allow 350 μl for eachadditional sample). Dilute DNA stock (100 μg/ml) 50-fold to give 2 μg/ml(20 μl added to 980 μl 1×TE). Prepare DNA standards as follows:

DNA Working Stock 1xTE Final Concentration DNA/ml 400 μl  0 μl 1000 ng 200 μl 200 μl 500 ng 100 μl 300 μl 250 ng  40 μl 360 μl 100 ng  20 μl380 μl  50 ng  10 μl 390 μl  25 ng  4 μl 396 μl  10 ng  0 μl 400 μl  0ng

Dilute samples 25× (16 μl sample+384 μl 1×TE). Add 100 μl of theappropriate standard to the wells of a 96-well plate or sample. Allstandards and samples to be assayed in triplicate. Add 100 μl ofPicoGreen solution to each well and incubate at RT for 2-3 min. Readplate on fluorescent plate reader. Excite at 485 nm and read at 538 nm.FIGS. 11 and 12, and the table below summarize the total in vitro DNAcontent.

Group PLL C CS CSF CSL CSL2 CSFL CSFL2 96.23984 863.0373 869.4763858.894 597.08 756.925 1472.216 2743.867 188.5894 558.7809 802.057884.0427 446.615 831.7972 1373.569 2074.91 96.8695 859.8953 597.4948566.9837 688.0478 687.7629 1242.719 1932.061 Avg. 127.2329 760.5711756.3427 769.9735 577.2476 758.8284 1362.835 2250.279 St. dev. 53.13723174.7625 141.6363 176.2435 121.9321 72.03604 115.1246 433.3854

The data above demonstrated that laminins and fibronectin are essentialin Schwann cell activity as well axonal growth. It was clear that thepresence of both laminin-1/-2 as well as fibronectin led to elevatedlevels of cell proliferation. To be sure, when both laminins were usedtogether in combination with fibronectin and collagen, the highestlevels of proliferation were seen.

Given the benefit of the above disclosure and description of exemplaryembodiments, it will be apparent to those skilled in the art thatnumerous alternative and different embodiments are possible in keepingwith the general principles of the invention disclosed here. Thoseskilled in this art will recognize that all such various modificationsand alternative embodiments are within the true scope and spirit of theinvention. The appended claims are intended to cover all suchmodifications and alternative embodiments. It should be understood thatthe use of a singular indefinite or definite article (e.g., “a,” “an,”“the,” etc.) in this disclosure and in the following claims follows thetraditional approach in patents of meaning “at least one” unless in aparticular instance it is clear from context that the term is intendedin that particular instance to mean specifically one and only one.Likewise, the term “comprising” is open ended, not excluding additionalitems, features, components, etc.

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What is claimed is:
 1. A method for making a nerve guide conduit with aninternal matrix comprising: providing an outer collagen tube wherein thetube is hollow; filling the collagen tube with a slurry comprisingcollagen, chondroitin sulfate, and one or more inductive macromolecules,freezing the slurry along an axial direction of the slurry, with theslurry having no detectable radial thermal gradient, to form a frozenslurry having parallel axially aligned crystals, freeze drying thecollagen tube and the frozen slurry to remove the parallel axiallyaligned crystals and form the internal matrix having parallel axiallyaligned internal matrix pores that span one end of the matrix to theother.
 2. The method of claim 1 wherein the inductive macromolecules areselected from the group consisting of fibronectin, laminin-1, andlaminin-2.
 3. The method of claim 1 wherein the step of freezingincludes contacting a proximal end of the collagen tube to a heat sinkwith the slurry freezing from a proximal end of the slurry to a distalend of the slurry to form the frozen slurry.
 4. The method of claim 1wherein the step of freezing the slurry comprises providing a coolinggradient in the axial direction of the slurry by rapid heat transferfrom the slurry to a cooling medium, wherein the cooling gradient hassubstantially no radial component.
 5. The method of claim 1 includingthe step of insulating the collagen tube to provide no detectable radialthermal gradient during the step of freezing the slurry.
 6. The methodof claim 1 wherein the collagen tube is placed in a mold which isinsulated.
 7. The method of claim 6 wherein the mold is a polystyreneblock.
 8. The method of claim 1 wherein the step of freezing the slurrycomprises rapid heat transfer from the slurry to a cooling mediumthrough a thermally conducting screw, plug, or pole contacting aproximal end of the collagen tube and the cooling medium, with thefreezing moving from a proximal end of the slurry to the distal end ofthe slurry.
 9. The method of claim 1 wherein the internal matrix poreshave an average diameter of about 10 μm to about 300 μm.
 10. The methodof claim 1 wherein the internal matrix pores have an average diameter ofabout 50 μm to about 80 μm.
 11. The method of claim 1 wherein theconduit is cylindrical.
 12. The method of claim 1 wherein the collagentube has an internal diameter from about 1.5 mm to about 5.0 mm.
 13. Themethod of claim 1 wherein the conduit has a length from about 1 cm toabout 15 cm.
 14. The method of claim 1 wherein the collagen tubecomprises pores with an average diameter of about 100 μm to about 200μm.
 15. The method of claim 1, wherein the collagen tube has anabluminal surface, wherein the abluminal surface has an irregularsurface pore structure.
 16. The method of claim 15, wherein theabluminal surface pores have an average diameter of about 20 μm to about200 μm.
 17. The method of claim 1 wherein the slurry further comprises aglycosaminoglycan selected from the group consisting of dermatansulfate, keratin sulfate, and hyaluronic acid.
 18. The method of claim 1wherein freezing the slurry is performed using liquid nitrogen or dryice.
 19. The method of claim 1 wherein the hollow collagen tubecomprises alkali-treated (AT) collagen.
 20. The method of claim 2wherein the chondroitin sulfate, fibronectin, laminin-1, and laminin-2have a dry weight basis in the ratio of about 1:1:1:1.
 21. The method ofclaim 2 wherein the chondroitin sulfate, fibronectin, laminin-1, andlaminin-2 each have a concentration of about 5 μg/ml.
 22. A method formaking a nerve guide conduit with an internal matrix comprising:providing an outer collagen tube wherein the tube is hollow; filling thecollagen tube with a slurry comprising collagen, chondroitin sulfate,and one or more inductive macromolecules selected from the groupconsisting of fibronectin, laminin-1, and laminin-2 or any combinationsthereof; freezing the slurry along an axial direction of the slurry,with the slurry having no detectable radial thermal gradient, to form afrozen slurry having parallel axially aligned crystals, freeze dryingthe collagen tube and the frozen slurry to remove the parallel axiallyaligned crystals and form the internal matrix having parallel axiallyaligned internal matrix pores that span one end of the matrix to theother, wherein the outer collagen tube with the internal matrix forms aconduit comprising a microenvironment that enhances Schwann cellsurvival.
 23. A method for making a nerve guide conduit with an internalmatrix comprising: providing an outer collagen tube wherein the tube ishollow; filling the collagen tube with a slurry comprising collagen,chondroitin sulfate, and one or more inductive macromolecules selectedfrom the group consisting of fibronectin, laminin-1, and laminin-2 orany combinations thereof; direct freeze-drying the collagen tube and theslurry along an axial direction of the slurry, with the slurry having nodetectable radial thermal gradient, to form a frozen slurry havingparallel axially aligned crystals, wherein the direct freeze-drying ofthe collagen tube and the slurry removes the parallel axially alignedcrystals and forms the internal matrix having parallel axially alignedinternal matrix pores that span one end of the matrix to the other, andwherein the outer collagen tube with the internal matrix forms a conduitcomprising a microenvironment that enhances Schwann cell survival.